The overall goal of this research is to gain insight into the mechanism of the regulation of protein synthesis initiation in animal cells. The rate of protein synthesis in animal cells is regulated by the status of the phosphorylation of the smallest alpha-subunit of eukaryotic initiation factor 2 (Eif2). p67, a cellular glycoprotein, protects eIF2alpha from phosphorylation by active kinases, PKR and HCR. The O-GlcNAc moieties of p67 are required for its activity. In response to stress such as viral infection, mitosis, and possibly others, p67 becomes inactive because of the high level of the activity of p67-deglycosylase which removes the O-GlcNAc moieties from p67. In this research the detailed structure-function relationship of p67 will be studied by genetic and biochemical analysis. Specific aims of this research are to identify and perform functional analyses for (i) p67 dimerization domain(s), (ii) p67 domains that are binding to eIF2alpha, eIF2gamma, PKR, and p67-DG, (iii) GlcNAc modification sites of p67, (iv) the phosphorylation site(s) where PKR transfers its phosphoryl group to p67, (v) the "lysine boxes" and an "acid box" of p67 in the regulation of protein synthesis, and (vi) the methionine aminopeptidase activity of rat p67. Further experimentations will be performed to understand the regulatory roles of p67 and p67-deglycosylase in the initiation of protein synthesis during cellular stress responses such as terminal differentiation, and viral infections. To obtain a comprehensive picture about the regulation of the p67 activity, p67-deglycosylase which is specific to p67, will be purified, cloned, and characterized. This research is expected to provide valuable information regarding the mechanism of protein synthesis initiation in normal, cancerous, and virus- infected animal cells.